Key result
The optimum poly(Y) tract length for trans splicing in T. brucei is approximately 25 nucleotides, and an AC dinucleotide at positions -3 and -4 can decrease trans splicing by 20-fold.
Identified sequence requirements for efficient RNA trans splicing in T. brucei, which can aid in computational identification of 3' splice sites and genome annotation.
May refine splice-site prediction in trypanosomes; leaves open in vivo validation and cross-species relevance.
mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5' splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts], 3' splice sites (3'SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3'SS, we constructed a luciferase-beta-galactosidase double-reporter system. By testing approximately 90 sequences, we demonstrated that the optimum poly(Y) tract length is approximately 25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3'SS is important, and an AC dinucleotide at positions -3 and -4 can lead to a 20-fold decrease in trans splicing. However, efficient trans splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3'SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome.
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Siegel et al. (2005) studied Trypanosoma brucei RNA trans splicing. Sequence motif variations (poly(Y) tract length/composition, spacer length/composition) vs. Wild-type or baseline sequence motifs was evaluated on trans-splicing efficiency (measured by relative luciferase activity). The optimum poly(Y) tract length for trans splicing in T. brucei is approximately 25 nucleotides, and an AC dinucleotide at positions -3 and -4 can decrease trans splicing by 20-fold.